Simultaneous Determination of α-Arbutin, β-Arbutin, Kojic Acid, Nicotinamide, Hydroquinone, Resorcinol, 4-Methoxyphenol, 4-Ethoxyphenol and Vitamin C from Skin Lightening Products by LC-UV

Skin lightening products have become increasingly in demand in recent years, especially in Asian markets. Skin lightening products are expected to whiten the skin as well as to tone skin or to treat pigmentation disorders such as freckles, melasma, pregnancy markers, and age spots [1]. Arbutin (α-arbutin and β-arbutin), kojic acid, nicotinamide and vitamin C are commonly used skin-whitening agents in cosmetic products. Several studies showed that these agents were tyrosinase inhibitors [2]. Resorcinol is used for removing hard, scaly, or roughened skin. Hydroquinone, 4-methoxyphenol and 4-ethoxyphenol have been banned in the European Union for the topical use in skin lightening application due to the high incidence of ochronosis and leukomelanodema.

Currently there is no analytical method for simultaneously determination of nine titled analytes in one matrix. In this study, an HPLC-UV method has been developed to determine α-arbutin, β-arbutin, kojic acid, nicotinamide, hydroquinone, resorcinol, 4-methoxyphenol, 4-ethoxyphenol and vitamin C from skin lightening products in a single run. The separation of nine compounds was achieved on a reverse phase C-18 column material within 30 minutes. The mobile phase was composed of water and methanol, both containing acetic acid. The column temperature was maintained at 45 °C. Stabilities of analytes at different pH range were estimated, and extraction method was validated. 20 mM NaH₂PO₄ in 10% MeOH (MeOH/water = 10:90, v/v) at pH 2.3 was proved to be the best solvent system for sample preparation. The method was validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). The developed HPLC-UV method was successfully applied on quantification of vitamin C, α-arbutin, β-arbutin, kojic acid, nicotinamide, hydroquinone, resorcinol, 4-methoxyphenol and 4-ethoxyphenol from 57 different skin lightening products obtained in the United States and India.

Fig. 1: HPLC-UV chromatograms of mixture of nine standards and some skin whitening products at UV 260nm [Vitamin C (1), β-Arbutin (2); α-arbutin (3), kojic acid (4), nicotinamide (5), hydroquinone (6), resorcinol (7), 4-methoxypheno (8) and 4-ethoxyphenol (9)]

Fig. 2: HPLC-UV chromatograms of mixture of nine standards and some skin whitening products at UV 280nm [Vitamin C (1), β-Arbutin (2); α-arbutin (3), kojic acid (4), nicotinamide (5), hydroquinone (6), resorcinol (7), 4-methoxypheno (8) and 4-ethoxyphenol (9)]

Acknowledgements: This research is supported in part by “Science Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 5U01FD004246, the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58 – 6408 – 02 – 1-612. References: [1] Thongchai W, Liawruangrath B, et al. (2007) J Cosmet Sci, 58: 35 – 44. [2] Lin JW, Chiang HM, et al. (2008) J Food Drug Anal, 16: 1 – 10.